In the eukaryotic nucleus, an energy source is required for remodeling chromatin at S phase and at M phase, and during the synthesis and export of RNA. Although nucleoside triphosphates fuel most processes in the cell, other high energy intermediates are involved. Prothymosin alpha, an abundant, acidic, nuclear protein, which in many systems and tissues is expressed in abundance only in growing cells, has been identified by our laboratory as energy-rich. We have demonstrated that this small, presumably unfolded, mammalian protein contains stoichiometric amounts of phosphorylated glutamic acid residues and that a specific glutamic acid rich peptide is the sole repository of the phosphate groups. To understand the function of prothymosin alpha in greater detail, the turnover of its high energy phosphates was examined in metabolically manipulated cells. Phosphate half-lives of 72-89 min were recorded in NIH3T3 cells in all stages of the cell cycle with the exception of M phase where a value of 174 min was observed in nocodazole- arrested cells. In the presence of actinomycin D, the value was about 145 min regardless of whether cells were quiescent or growing. In these experiments, reduced utilization of prothymosin alphas glutamyl phosphates signalled by an increase in their half-lives accompanied the attenuation or abolition of transcription. Prothymosin alpha was also examined during apoptosis in HeLa cells with the following results: prothymosin alpha was cleaved near the C- terminus by a caspase 3-like enzyme which removed 10-14 residues including the core nuclear localization signal; the truncated protein was no longer confined to the nucleus under conditions in which the intact protein was retained; the truncated protein was deficient in phosphate. These data suggest that egress of prothymosin alpha from the nucleus and entry into it occur continuously in normal cells and that in apoptotic cells re-entry is precluded. Using imaging techniques, together with methods to perturb the metabolism of NIH3T3, COS-1, and HeLa cells, we now have evidence that prothymosin alpha shuttles between the nuclear and cytoplasmic compartments. A probable function has emerged; prothymosin alpha assists in the movement of macromolecules, as well as small charged molecules, through channels in the nucleus and the nuclear envelope probably by virtue of its extraordinary negative charge. Prothymosin alpha appears to be a mobile salt packet used to facilitate nuclear events among which is transcription. Although the high energy phosphates are apparently essential for the function of prothymosin alpha, the mechanism by which they participate has yet to be determined. - Mammalian Cell, Nucleus, Phosphorylation, proliferation, prothymosin alpha,